This program is extended from our continuing efforts to investigate the immune response to the hepatitis C virus (HCV) in both humans and experimental animals. In extensive earlier studies, we identified immunodominant and neutralizing epitopes on the hepatitis C virus that will now be further investigated to examine the relationship between immune response and persistent infection. This long-term project can be globally subdivided into four phases. The initial phase is design and construction of immunogenic plasmids. This includes monitoring the development and course of HCV cellular and humoral immunity and selecting the most effective gene delivery systems. The second phase is to determine the immunogenicty of these constructs. During this step, we are evaluating a variety of gene delivery strategies and the use of adjuvants and other immune modifiers. The third phase is to evaluate the immune responses and the final phase is to conduct protectivity studies in chimpanzees. Great efforts have been devoted to evaluating HCV immune response including assay development for measurting cell mediated immunity in mice, chimpanzees and humans. The protectivity study would have to be conducted in an appropriate animal model. Each of these phases have many different components and the development, implementation, evaluation and improvement steps could be over-lapping. In FY 95 and FY 96, we initiated a new project to examine the potential of nucleic acid vaccination for the prevention and/or treatment of HCV infection. The long-term goal of both the basic immunology studies and the DNA vaccine studies is to develop models for immune therapy of chronic viral infections of the liver. One of the advantages of genetic immunization is that the endogenously expressed proteins can be recognized by class I MHC molecules and expressed on the cell surface. The MHC-antigen complex on the cell surface can be recognized by cytotoxic T lymphocytes (CTL), which, in turn, are activated and attack infected cells. The possibility of inducing an immune response to HCV core protein using DNA immunization provides an attractive alternative to classic vaccination. There are many problematic issues related to the vaccine development for hepatitis C. One major concern is the genetic instability of the infectious agent. There are two hypervariable regions in the putative HCV envelope proteins. Immune escape mutants have been identified in these regions. Experimentally infected chimpanzees and HCV-infected patients have been found to repeat bouts of infection with either homologous or new strains of HCV. This failure to develop protective immunity links to the high chronicity rate in HCV infection. Directly inducing strong cell-mediated immunity, especially protective cytotoxic T lymphocyte responses, may not only help in preventing initial HCV infection, but may serve as a mechanism for immune modulation to overcome existing infection. Using the mouse model, we were able to evaluate the induction of antibodies to several different plasmid constructs containing both HCV structural and non-structural genes. We were also able to develop assays to measure both humoral and cell-mediated immune responses, including CTL activities, in the mouse model. In the past years, we have tested the genetic sequences of many HCV-related immunogens to establish the best candidate DNA vaccine. We have also studied methods of vaccine delivery and immunity augmentation procedures; accumulated extensive experience in measuring humoral and cell-mediated immunity; and developed effective immunization strategies in small experimental animals. During last period, we have conducted studies combining several immunogens to evaluate their interaction or interference. We also developed a protein boost strategy. After recognizing the induction of potent cellular and humoral responses in small animals using our DNA prime-protein boost procedurrres and formulations, we initiated a protope multicomponent (core, envelope, NS3, and NS5b) HCV vaccine trial in chimpanzee animal model in FY 2004. The chimpanzee experiment is still ongoing. The early results indicate that our immunization strategy can induce strong HCV specific CD4+ and CD8+ T-cell immunity, as well as antibody activities in chimpanzee. After challenging two immunized chimpanzees with homologous HCV virus, both animals became infected. However, one animal resolved the infection nine weeks later and remained virus free for 18 months. A protocol for this chimpanzee has been initiated to investigate the mechanism of protective immunity through serial homologous and hetrologous virus challenges to this animal. The other animal is shown to be chronically infected for more than a year with consistently positive viral load. A protocol for this chronically infected chimpanzee has been undertaken to demonstrate possible immune therapy through various immunization regimens augmenting the immunity to overcome the infection.[unreadable] [unreadable] During the last year, we applied techniques and reagents developed during this study: ELISPOT (Enzyme Linked Immune SPOT assay) to evaluate HCV infected patient antibody producing cells. The postdoctoral fellow who worked on this project may extend this study to earlier infected patients found in Japan. A collaborative project may be developed.